P53 R273H Mutation Promotes Ionizing Radiation-induced Epithelial-mesenchymal Transition In Lung Cancer Cells

Lung cancer is one of the most common cancers,and its morbidity and mortality have always been high,of which about 80%are non-small cell lung cancer（NSCLC）.P53 is one of the genes with the highest mutation rate in non-small cell lung cancer,with a mutation rate of about 50%,of which R273H mutation is the most common.After mutation of p53 gene（mut-p53）,its tumor suppressor function is lost,and a new function that is different from wild-type p53（wt-p53）can be obtained,which is defined as gain of function（GOF）.This GOF can promote tumor malignant progression,including increased lung cancer cell invasion and metastasis.Radiotherapy is a conventional method of lung cancer treatment,which can inhibit the growth of lung cancer cells and reduce the recurrence rate of lung cancer.The side effects of radiotherapy cannot be ignored.After radiation,fibroblasts are transformed into myofibroblasts,which lead to the remodeling of lung structure.Fibroblasts differentiate into myofibroblasts,called fibroblasts or epithelial cells from the epithelial-mesenchymal transition（EMT）.EMT is a cellular process in which cells lose their epithelial characteristics and acquire mesenchymal characteristics.EMT is associated with multiple tumor functions,including tumor malignant progression,tumor cell migration,metastasis,and resistance to therapy,and is closely related to tumor initiation,invasion,and metastasis.However,the mechanism of radiation-induced EMT in lung cancer cells is still unclear.Early growth response factor 1（EGR1）is a transcription factor that is mainly involved in tissue damage,immune response and fibrosis.EGR1is closely related to the occurrence and development of cancer,and may be involved in tumor cell proliferation,invasion,metastasis and tumor angiogenesis.Studies have shown that mut-p53 protein can bind to the EGR1 promoter region and promote the expression of EGR1,but whether this mechanism is involved in the occurrence of radiation-induced EMT in lung cancer cells remains to be further elucidated.Purpose:By constructing a cell model stably expressing wt-p53 and mut-p53 in H1299 cells with p53 gene deletion,we observed the effect of R273H mutant p53 on the EMT phenotype of H1299 cells under the action of ionizing radiation;observed the stable expression of H1299-p53^R273H Changes in the expression of EGR1 in cells,to explore the regulatory effect of p53^R273H on EGR1,and to clarify the important role of p53^R273Hin ionizing radiation-induced EMT in lung cancer cells.Method:1.The A549 cells and H1299 cells were irradiated with X-ray doses of 0,6,8,and10 Gy respectively,and the cells were collected at 0,24,and 48 hours after irradiation,and the effects of radiation on the EMT markers of A549 and H1299 cells were observed.2.Scratch and Transwell test methods were used to observe the migration and invasion ability of A549 and H1299 cells and the changes of the migration and invasion ability of H1299 cells after stable transfection.3.Using lentivirus transfection method,p Lenti-CMV-p53 and p Lenti-CMV-p53（R237H）were stably transfected into H1299 cells to construct H1299-p53^WT and H1299-p53^R273H cell models.4.The cell cycle changes of H1299-p53^WT and H1299-p53^R273Hwere detected by flow cytometry.5.The expression changes of EMT markers,p53 and EGR1 were detected by Western Blot and q-PCR.6.To verify the regulatory relationship between p53 and EGR1 by CHIP assay.Result:1.Radiation-induced EMT in A549 cellsA549 cells were irradiated with 0,6,8,and 10 GyX-rays,and the changes of intracellular EMT markers were detected at 0,24,and 48 h.The results showed that,compared with the control group,the expression of E-cadherin protein showed a downward trend after 8GyX-ray irradiation,and the most significant decrease was at48h after radiation;the protein expression levels of N-cadherin and Vimentin showed an upward trend,and were up-regulated at 48h after radiation.Most obvious.The changes of EMT markers in H1299 cells were detected under the same conditions,and the results showed that the protein expression levels of E-cadherin,N-cadherin and Vimentin did not change significantly with the increase of radiation dose and time.2.Irradiation-induced changes in the migration and invasion abilities of A549 cells and H1299 cellsAfter 8GyX-ray irradiation of A549 cells for 24 and 48h,compared with the control group,the distance of cell migration increased significantly（p<0.01）,suggesting that ionizing radiation can increase the migration ability of A549 cells;after8GyX-ray irradiation of H1299 cells for 24 and 48h,the cell migration distance did not change significantly.After 8Gy irradiation of A549 and H1299 cells for 24h,compared with the control group,the number of A549 cells in the lower chamber was significantly increased（p<0.01）,while the H1299 cells showed no significant change compared with the control group.3.Construction of H1299-p53^WTand H1299-p53^R273H cell modelsThe p53^WT and p53^R273H plasmids were transferred into H1299 cells by lentiviral transfection.Compared with the control group,the m RNA and protein levels of p53were significantly increased in the H1299-p53^WT and H1299-p53^R273Hcell models（p<0.001）;and It was observed that H1299-p53^WT cells were round,while H1299-p53^R273H cells were spindle-shaped.Cytoskeleton staining showed increased microfilaments and pseudopodia,showing mesenchymal cell morphology.4.Irradiation-induced EMT phenotype changes in H1299-p53^WT and H1299-p53^R273HcellsAfter 8GyX-ray irradiation for 48h,compared with the control group,the expression of E-cadherin protein in H1299-p53^WT and H1299-p53^R273Hcells was significantly decreased,while the protein expression of N-cadherin and Vimentin was significantly increased;Compared with the control group,the migration distance of H1299-p53^R273H cells was significantly increased（p<0.001）,and the cell invasion ability was also significantly enhanced（p<0.0001）.Compared with H1299-p53^WT cells,H1299-p53^R273H had significantly enhanced cell migration and invasion abilities（p<0.001）;after 8GyX-ray irradiation,H1299-p53^WT cells had a reduced proliferation ability（p<0.001）,while H1299-p53^R273H cells proliferated Enhanced ability（p<0.001）.5.The effect of radiation on the expression of p53 and EGR1 in H1299-p53^WT and H1299-p53^R273HcellsAfter 8GyX-ray irradiation for 48h,compared with H1299-p53^WT cells,the protein expression levels of p53 and EGR1 in H1299-p53^R273H cells were significantly increased,while the expressions of p53 and EGR1 in H1299-p53^WT cells did not change significantly;IF results showed that EGR1 expression was increased in H1299-p53^R273H cells and mainly accumulated in the nucleus.6.The effect of p53^R273H on the binding of p53 to the EGR1 promoter regionBy comparing the two cell models of H1299-p53^WTand H1299-p53^R273H,we found that the binding of p53^R273H to the EGR1 promoter region was significantly increased in H1299-p53^R273H cells.Conclusion:1.P53 is involved in radiation-induced EMT in lung cancer cells.2.Irradiation can induce increased expression of p53 and EGR1 in H1299-p53^R273H cells,and increased the ability of invasion,migration and proliferation of H1299-p53^R273Hcells.3.Irradiation can induce the enrichment of p53^R273H in the EGR1 promoter region and promote the expression of EGR1.